Cytotoxic T lymphocytes (CTLs) have crucial roles in eliminating host cells that contain intracellular pathogens and those that have undergone malignant transformation (Doherty and Christensen (2000) Annu. Rev. Immunol. 18: 561-592). In the past three decades, the 51Cr-release assay has been used to quantify antigen-specific cell-mediated cytotoxicity activity (Brunner et al. (1968) Immunology 14: 181-196). In this assay, target cells labeled with radioactive 51Cr are incubated with effector cells for 4-6 hours. Target-cell death is then measured by detecting radioactivity released into the culture supernatant.
Although relatively reproducible and simple, this assay has numerous disadvantages (Doherty and Christensen (2000) Annu. Rev. Immunol. 18: 561-592). First, bulk cell-mediated cytotoxicity activity is measured using ‘lytic unit’ calculations that do not quantify target-cell death at the single-cell level. Second, CTL killing of primary host target cells often cannot be studied directly as only certain types of cells, primarily immortalized cell lines, can be efficiently labeled with 51Cr (Nociari et al. (1998) J. Immunol. Meth. 213: 157-167). Third, target-cell death is measured at the end point of the entire process and thus provides little information about the kinetic interaction of effectors and targets at the molecular and cellular levels. Fourth, the radio active conventional assay using chromium 51 isotope (51Cr) results in a very large background (noise) signal due to a large amount of spontaneous nonspecific release of the isotope from the target cells and often very heterogeneous loading of the isotope in the selected target cells. Fifth, the amount of released radio activity is therefore not a direct measure of cell death but rather membrane permeability change and spontaneous release of the isotope from the loaded cells due to processes other than the cellular cytotoxicity brought about by the cytotoxic effector cells. Consequently, the conventional chromium release assay has difficulty in detecting definite but less potent cytotoxic effects, i.e., it is difficult to distinguish a signal caused by cell-mediated cytotoxic activity from the assay's background radioactivity. Measurement of 51Cr release does not permit monitoring the physiology or fate of effector cells as they initiate and execute the killing process. Finally, radioactive materials require special licensing and handling, which substantially increases cost and complexity of the assay.
More recently developed immunologic methods, including major histocompatibility complex (MHC)-tetramers, intracellular cytokine detection and ELISPOT assays, have greatly improved sensitivity to enumerate antigen-specific T cells; however, these newer methods do not assess the cytolytic function of antigen-specific cell-mediated cytotoxicity (Altman et al. (1996) Science, 274: 94-96 (1996); erratum: 280: 1821 (1998); Butz and Bevan (1998) Immunity 8:167-175; Maino and Picker (1998) Cytometry 34: 207-215). Given emerging data indicating that antigen-specific CD8+ T cells may be present in certain chronic infections or malignancies, but blocked in their ability to lyse target cells, assays that measure all the effector cell functions at the single-cell level are needed (Appay et al. (2000) J. Exp. Med. 192: 63-75; Lee et al. (1999) Nature Med. 5: 677-685; Zajac et al. (1998) J. Exp. Med. 188: 2205-2213).
In recent efforts to overcome some of the limitations of the 51Cr-release assay through development of flow cytometry based cell-mediated cytotoxicity assays, some groups have measured target-cell death based on the amount of fluorochrome released from or retained in the prelabeled target cells (Sheehy et al. (2001) J. Immunol. Meth. 249: 99-110; erratum: 252: 219-220 (2001)), or detected the late stages of target-cell death using intercalative DNA dyes (Lecoeur et al. (2001) J. Immunol. Meth. 253: 177-187). However, none of these assays reveal the fundamental processes responsible for the initiation and execution of target-cell killing, and none have yet been applied to analyses of primary cell-mediated cytotoxicity generated in vivo following antigenic exposure.